The immobilization of active proteins at interfaces is a challenge common to many areas of applied biological technology. For example, immobilization of proteins at interfaces is required for diagnostic systems, such as immuno assays. In addition, proteins are attached to bio-compatible polymer for delivery into biological systems for diagnostic and therapeutic applications. Immobilization of proteins upon an interface is also required for the continuing development of ultra-sensitive instrumentation based on optical tweezers and scanned tip microscopes for probing the activities and mechanical properties of single protein molecules.
Any attachment of a protein for immobilization would ideally minimize structural perturbation and maximize protein activity, allow immobilization of proteins with a controlled orientation to the surface, and provide a consistent linkage compliance to the surface. This is important for accurate and consistent results when measuring the properties of a particular protein. Other properties of an ideal immobilization scheme are preservation of biomolecule activity, specificity, high binding stability, reversible binding under non-denaturing conditions, controlled immobilization density, and convenient co-immobilization of multiple proteins, or other molecules. Likewise, in complexes of a protein and a biomolecule that are not necessarily used in coatings, perturbation of the structure of the protein should be minimized, and the maximum protein activity be preserved.
Several approaches using chelated metal ions have been reported that allow histidine-tagged proteins to be immobilized at several types of interfaces, such as lipid interfaces and lipid monolayers with metal-chelating lipids, gold surfaces with self-assembling monolayers formed with metal-chelating alkanethiols, and oxide surfaces with metal-chelating silanes. A problem with these methods is that they are complex and not convenient to use.
U.S. Pat. No. 5,674,677 to Peterson describes a method for joining two amino acid sequences by coupling an organic chelator to an protein, e.g., an enzyme, and charging the chelator with a metal ion. This complex is then mixed with any protein containing a histidine tag to couple the complex with the histidine tagged protein.
So-called PLURONIC.TM. surfactants shall be referred to herein as `P-surfactants`. P surfactants are poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO) block copolymers, and have been shown to be very passive with respect to protein activity. Certain P-surfactants have little or no affinity for proteins and form self-organized coatings on hydrophobic surfaces to provide a passive, non-active surface with respect to proteins. U.S. Pat. No. 5,516,703, to Caldwell et al. (Caldwell et al.), which is hereby incorporated by reference, describes a method for introducing reactive end groups to a P-surfactant, and chemically coupling to proteins through chemical covalent bonds with the reactive end group. The P-surfactant with the reactive end groups is adsorbed upon a surface, and then reacted to couple to a protein to immobilize the protein upon the surface.
The Caldwell et al. system is a successful method for immobilizing a protein on a surface that preserves the activity of the protein, and that prevents non-specific adsorption of proteins. However, the Caldwell et al. system has several limitations. Attachment to the P-surfactant must be through chemical reaction to form a covalent bond, and certain proteins do not lend themselves well to such attachment. For example, a preferred attachment in Caldwell et al. is through disulfide groups, which attach to thiol groups in amino acids. A particular protein may not have thiol groups, or the thiol groups may not be present in a suitable position in the protein for attachment. In addition, thiol groups may exist at multiple places in the protein, which allows attachment to the P-surfactant molecule at anyone or more of several locations. The result can distort the natural conformation of the protein, which can adversely affect its activity. In addition, the protein may not attach consistently and predictably where several thiol groups may be present.